The first field improved upon by the present invention is the field of nucleic acid diagnostics. The field of nucleic acid diagnostics uses the tools of molecular biology to detect the presence of bacterial and viral infectious agents, genetic variation, and diseases such as cancer. One of the first steps in nucleic acid diagnostics is generally the amplification of one or more target nucleic acid sequences specific for the biological entity of interest in one or more samples. This amplification step provides the sensitivity to detect very small numbers of nucleic acid molecules containing the target sequence(s). Several nucleic acid amplification methods have been developed, including the well-known polymerase chain reaction (PCR) as well as the ligase chain reaction (LCR) and the self-sustained sequence replication (3SR) method. The specificity of these amplification methods relies on the use of oligonucleotide primer sets which uniquely identify a particular target nucleic acid sequence. This specificity can be very high; for example, in multiplex PCR, different primer sets are used to amplify separate target sequences within the same nucleic acid molecule in a single tube.
Following the amplification step, the presence of a particular amplified target nucleic acid sequence is then determined by a variety of ways. One of the most common methods is direct visualization of the desired product by electrophoresing an aliquot of the amplification reaction in an agarose or acrylamide gel. Since the amplification products are separated on the basis of size, this detection method is precluded if discrimination between different amplification products, i.e., different target sequences, of about the same size is desired.
More sensitive detection methods involve hybridization of the amplified products with an oligonucleotide (oligo) probe having a sequence complementary to a portion of the target sequence of interest followed by detection of the oligo-target hybrid. These hybridization methods generally include immobilization of the amplified products or immobilization of the oligonucleotide probe to a solid support, wherein either the oligo or the target nucleic acid bears a detectable label. For example, T. R. Gingeras et al., "A Transcription-Based Amplification System" in PCR Protocols: A Guide to Method and Applications (Michael A. Innis et al. eds., 1990), pp. 245-252, herein incorporated by reference, disclose a bead-based sandwich hybridization method in which sephacryl beads containing an immobilized oligonucleotide specific for a segment of the target sequence were used to capture amplified products that had hybridized in solution to a .sup.32 P-labeled detection oligonucleotide specific for a different segment of the target sequence. The presence of the target sequence was detected by assaying the beads for radioactivity.
Other solid supports may be used to immobilize one of the hybridization partners. For example, in the well-known dot-blot method, aliquots of the amplified sample are dotted on a nylon membrane which is then probed with a labeled oligonucleotide. If detection of multiple target nucleic acid sequences is desired, this dot blot method can be quite time consuming in that for n target sequences, the membrane must be stripped between sequential hybridization with each of n probes, or alternatively, each amplified sample must be immobilized on n membranes and each membrane hybridized to one of n probes. This problem is addressed by the "reverse-dot blot" method disclosed by H. A. Erlich and T. L. Bugawan, "HLA DNA Typing" in PCR Protocols: A Guide to Method and Applications (Michael A. Innis et al. eds., 1990), pp. 261-271, herein incorporated by reference, in which the amplified product, labeled during amplification, is hybridized to an immobilized array of oligonucleotide probes.
Recently developed hybridization-based nucleic acid detection methods do not rely on amplification of a target sequence for sensitivity, but instead amplify the signal from each immobilized oligo-target hybrid. Such signal amplification methods include the branched DNA (bDNA) probe technology developed by Chiron (Emeryville, Calif.).
While the above-described reverse dot blot method allows for the simultaneous detection of multiple nucleic acid sequences, this method is not readily adaptable to economical large scale use or automation. For example, the production of multiple units, i.e., membranes containing duplicate arrays of oligonucleotide probes, requires precision spotting of equivalent amounts of probes to defined areas of replicate membranes in a sequential fashion. Moreover, performing detection of a large number of sequences with the reverse dot blot method requires covering a fairly large membrane surface area with hybridization reagents, some of which are expensive.
However, in many situations it would be advantageous to rapidly screen a sample for the presence of multiple nucleic acids simultaneously using a method that is economical to perform and amenable to automation. For example, in the biomedical field, it would be beneficial to have a technique to rapidly and economically screen donated blood from multiple individuals for different known pathogenic viruses. Also, due to the increasing concern about the potential use of biological weapons by terrorists and by armies in war, the ability to rapidly screen multiple air and water samples in the field for nucleic acids of known and/or unknown sequence could improve the ability to treat both civilian populations and military personnel exposed to such weapons. Finally, an optimal method for simultaneous detection of multiple nucleic acids would be capable of directly detecting small numbers of target nucleic acid sequences by increasing the hybridization signal. Such detection would lend the capability of detecting in a sample the presence of single pathogenic microorganisms which carry low copy numbers of the target nucleic acid sequence.
The second field improved by the present invention is the field of immunodiagnostics. Enzyme linked immunoassay, commonly referred to as ELISA, is a well known technique for the detection and measurement of antigens or antibodies in solution which uses enzyme-linked antigens or antibodies to detect an antigen-antibody reaction. This technique has been used in a variety of immunodiagnostic applications such as seriodiagnostics to detect antigens from a wide range of specific viruses, bacteria, fungi, and parasites, and to measure the presence of antibodies against these various microorganisms. ELISA is also used to monitor factors involved in noninfectious diseases such as hormone levels, hematological factors, serum tumor markers, drug levels, and antibodies.
Typically, the enzyme used in ELISA is selected from alkaline phosphatase, horseradish peroxidase, and beta-galactosidase and it is coupled to an antibody or antigen. The binding of the enzyme-linked antibody or antigen to its corresponding antigen or antibody is detected by adding substrates that, upon reaction with the enzyme, are converted into colored reaction products or give off luminescence.
An antigen-antibody reaction is traditionally defined as the interaction between an antigenic determinant, or epitope, on the antigen molecule and a corresponding antigen-combining site, or paratope, on the variable region of the antibody molecule. In addition to this interaction, the paratope on the antibody molecule may also serve as an antigenic site, i.e., be recognized by a paratope on the variable region of a second antibody molecule. In this situation, the paratope on the first antibody is referred to as the idiotope. Thus, in its broadest sense, the ELISA technique may be understood as capable of detecting the interaction of specific binding partners, or ligands, which include epitope-paratope interactions and idiotope-paratope interactions.
The type of binding partner interactions, which can be detected by ELISA, is partially illustrated by a recent description of a modified ELISA technique used to detect the presence of anti-HIV antibody in solution. Brennan et al., in Protein Engineering 7(4): 509-514 (1994), describes a modified alkaline phosphatase containing an epitope from the HIV-1 gp120 protein inserted onto its surface in the vicinity of the enzyme's active site. The activity of this modified alkaline phosphatase, which is comparable to the activity of wild type alkaline phosphatase, was reduced by almost half by the binding of an anti-gp120 monoclonal antibody to the gp120 epitope on the modified alkaline phosphatase.
One of the limitations of conventional ELISA methods is that they detect, in a linear or sequential fashion, only one type of antigen or antibody at a time. However, in many situations it would be advantageous to rapidly screen a sample for the presence of multiple antigens or antibodies simultaneously. For example, in the biomedical field, it would be beneficial to have a technique to rapidly determine which pathogen(s) of a number of possibilities has infected a patient so that an appropriate treatment can be implemented without the delay of additional screening. In addition, with the increasing concern about the potential use of biological and chemical weapons by terrorists against civilian populations and by armies in war as weapons of mass destruction, the ability to rapidly detect and identify multiple biological, chemical or toxin agents in the field could improve the ability to treat both civilian populations and military personnel exposed to such agents.
The third field improved by the present invention is the field of automated workstations. Such workstations are designed to automatically carry out sequential chemical reactions such as the amplification of DNA using the polymerase chain reaction.
State of the art automated workstations used in carrying out the polymerase chain reaction, such as Perkin-Elmer's ABI PRISM 877 integrated thermal cycler, automatically handle the cycling protocol. This cycling protocol consists of a standard multi-well thermal cycler integrated with a high-precision robot which conducts the pipetting of PCR primers, DNA polymerase, buffer, and other PCR reagents into individual tubes placed in the wells of the thermal cycler and the subsequent removal of the reaction tubes. The benefits of such an automated system is that the system frees researchers and lab assistants from manually pipetting the reagents into the individual reaction tubes and speeds the processing of samples by the use of multiple reaction tubes. In addition, such automated integrated thermal cycler systems reduce the chance for contamination from ubiquitous oligonucleotides which are found on almost every surface. Nonetheless, automated workstations work in a relatively slow step-wise fashion related in a large part to the physical constraints placed upon the robotic arm, which controls the automatic pipetting of aliquot and the subsequent removal and replacement of the reaction tubes. In addition, the movement of the robotic arm and the pipetting of PCR reagents into the reaction tubes still permits contamination of the reaction mixture by foreign oligonucleotides that might come in contact with the pipette orifice or be introduced by airborne particles. It would be very advantageous in the field of automated PCR workstations to have an apparatus which is capable of simultaneous amplification of oligonucleotides found in multiple samples and which eliminates the chance of contamination of the samples with stray oligonucleotides.
No related method, composition, or apparatus is known that uses a unique reporter molecule to identify the presence of the target antigen or target nucleic acid so as to permit the simultaneous detection of multiple nucleic acid sequences and/or multiple antigens from a biological sample and, wherein the sample containing the reporter molecule is continuously and automatically amplified in a closed apparatus before the ultimate detection of the presence of the reporter molecule is accomplished using electrochemiluminescent technology. Electrochemiluminescent technology is available from IGEN, Inc. of Gaithersburg, Maryland and is disclosed in detail in U.S. Pat. Nos. 5,310,687; 5,221,605; 5,238,808 and 5,147,806 which are herein incorporated by reference.